Application of walnut peptide in improving abundance of ackermania muciniphila in intestinal flora

Abstract

The invention discloses application of walnut peptide in improving abundance of ackermania muciniphila in intestinal flora, and relates to the technical field of functional peptide. The walnut peptide prepared by the method disclosed by the invention is mainly used for improving cognitive function decline caused by natural aging by regulating ackermania muciniphila in intestinal flora. Specifically, (1) the spatial memory function of a naturally aged mouse is obviously improved; (2) the diversity of intestinal microorganisms of naturally aged rats is recovered; (3) reversing the F / B ratio to remodel the intestinal microenvironment; and (4) the abundance of Ackermania muciniphila is obviously increased.

Description

Technical Field

[0001] The present invention relates to the technical field of functional peptides, and more specifically, to the application of walnut peptides in increasing the abundance of Akkermansia muciniphila in the intestinal flora. Background Art

[0002] With the acceleration of the global aging process, the incidence of cognitive function decline and related neurodegenerative diseases has gradually increased, becoming a major challenge to global public health. Alzheimer's disease (AD), as the most common neurodegenerative disease, not only seriously affects the quality of life of patients but also brings a heavy burden to society and families. At present, the main drugs for treating neurodegenerative diseases include rivastigmine, donepezil, and galantamine, etc., but these drugs are often accompanied by adverse reactions such as nausea, diarrhea, and vomiting. Therefore, the development of new treatment strategies, especially intervention methods based on natural bioactive substances, has become a research hotspot.

[0003] The pathogenesis of cognitive impairment is complex. In recent years, studies have shown that intestinal flora dysbiosis also plays an important role in the occurrence and development of cognitive impairment. The "gut-brain axis" is the key pathway connecting the gut microbiota and brain function. The gut microbiota can affect central nervous system function through multiple mechanisms such as regulating immune responses, metabolites, and neurotransmitters. There is evidence that the disorder of the intestinal flora can lead to impaired intestinal barrier function and low-grade chronic inflammation, thus exacerbating the decline of cognitive function. Akkermansia muciniphila (Akk), as an important intestinal microorganism, is outstanding in maintaining intestinal mucosal barrier function and alleviating intestinal inflammation. Supplementing Akk bacteria helps to improve cognitive impairment and repair the intestinal barrier at the same time. These findings reveal the potential role of regulating the intestinal microecology in improving cognitive function.

[0004] Therefore, the development of natural bioactive substances with good safety, which can regulate intestinal homeostasis to improve cognitive function, has important research value and broad application prospects. Summary of the Invention

[0005] In view of this, the present invention provides the application of walnut peptides in increasing the abundance of Akkermansia muciniphila in the intestinal flora.

[0006] To achieve the above object, the present invention adopts the following technical scheme:

[0007] The application of walnut peptides in increasing the abundance of Akkermansia muciniphila in the intestinal flora, and the preparation method of the walnut peptides with the regulatory effect on Akkermansia muciniphila includes the following steps:

[0008] (1) Extraction of walnut protein powder: Extract walnut protein powder from walnut meal by the alkali solution acid precipitation method;

[0009] (2) Preparation of walnut peptide: Stir and mix walnut protein powder with deionized water evenly; Pretreat in a water bath at 90 °C for 30 min; Under the conditions of pH 7.5 and 50 °C, use a composite plant hydrolase and pancreatic enzyme to carry out enzymatic hydrolysis for 12 h; Treat the obtained enzymatic hydrolysate at 95 °C for 10 min to inactivate the enzyme; After the enzymatic hydrolysate is cooled to room temperature, immediately centrifuge for 20 min; Collect the supernatant and freeze-dry to obtain walnut protein peptide.

[0010] Preferably, the stirring and mixing in step (2) is magnetic stirring, and the ratio of walnut protein powder to deionized water is 1:8.

[0011] Preferably, the addition amount of the plant protein hydrolase and pancreatic enzyme in step (2) is 0.6% of the mass of the walnut protein powder.

[0012] Preferably, the centrifugation parameters of the enzymatic hydrolysate in step (2) are 8000 rpm and 4 °C.

[0013] Preferably, the freeze-drying parameters in step (2) are a cold trap temperature of -45 to -50 °C, a pressure of 30 - 100 Pa, a freeze-drying time of 60 - 72 h, and the moisture content of the walnut peptide after freeze-drying is ≤ 7%.

[0014] More preferably, step (1) is specifically to grind walnut meal into powder, add 8 times the mass of deionized water and mix evenly, adjust the pH to 8.0, and stir at room temperature for 4 hours; Then centrifuge at 4 °C and 8000 rpm for 20 minutes, collect the supernatant and adjust the pH to 4.5, continue to stir at room temperature for 4 hours, and then centrifuge at 4 °C and 8000 rpm for 20 minutes again to collect the precipitate; Dissolve the precipitate in water at a solid-liquid ratio of 1:4, put it into a dialysis bag (cut-off molecular weight 8000 - 14000 Da), and dialyze with ultrapure water at 4 °C for 2 days to desalt, changing the water every 4 hours; Finally, freeze-dry the dialysate under the conditions of a cold trap temperature of -45 to -50 °C, a pressure of 30 - 100 Pa, and a freeze-drying time of 60 - 72 h to obtain walnut protein powder.

[0015] Beneficial effects:

[0016] The walnut peptide prepared by the method of the present invention plays an improving role in the cognitive function decline caused by natural aging by regulating Akkermansia muciniphila in the intestinal flora. Specifically manifested as: (1) Significantly improving the spatial memory function of naturally aged mice; (2) Restoring the intestinal microbial diversity of naturally aged mice; (3) Reversing the F / B ratio to reshape the intestinal microenvironment; (4) Significantly increasing the abundance of Akkermansia muciniphila.

[0017] The walnut peptide of the present invention has a good improving effect on the cognitive and memory disorders caused by natural aging and can be applied to the preparation of memory-improving products.

[0018] The present invention provides a new raw material basis and technical support for the development of products for improving cognition / promoting intestinal health, and has broad market application value.

[0019] Compared with synthetic drugs, the walnut peptides of the present invention have the advantages of excellent biological activity, low toxicity, and easy metabolism in the body for small molecule peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0021] Figure 1 It is a representative trajectory diagram of the Morris water maze place navigation test.

[0022] Figure 2 It is the escape latency of mice in the Morris water maze place navigation test.

[0023] Figure 3 It is the ratio of the moving distance of mice in the target quadrant to the total moving distance in the Morris water maze spatial exploration test.

[0024] Figure 4 It is the number of times mice cross the platform in the Morris water maze spatial exploration test.

[0025] Figure 5 It is a typical representative trajectory diagram of the Barnes maze place navigation test.

[0026] Figure 6 It is the escape latency of mice in the Barnes maze place navigation test.

[0027] Figure 7 It is the number of times mice explore the target hole in the Barnes maze spatial exploration test.

[0028] Figure 8 It is the α-diversity of the intestinal flora of mice.

[0029] Figure 9 It is the β-diversity of the intestinal flora of mice.

[0030] Figure 10 It is the ratio (F / B) of Firmicutes and Bacteroidetes in the intestinal flora of mice.

[0031] Figure 11 It is a column chart of the LDA value distribution of significantly different species in the intestinal flora of mice.

[0032] Figure 12 is the relative abundance of Akkermansia muciniphila in the mouse intestine. Detailed implementation manners

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] Example 1

[0035] A preparation method of walnut peptide

[0036] (1) Extraction of walnut protein powder: Extract walnut protein from walnut meal by the alkali solution acid precipitation method. First, grind the walnut meal into powder, add 8 times its mass of deionized water and mix evenly (magnetic stirring). After adjusting the pH to 8.0, stir at room temperature (15 - 35 °C) for 4 hours. Then centrifuge at 4 °C and 8000 rpm for 20 minutes, collect the supernatant and adjust the pH to 4.5, continue to stir at room temperature for 4 hours, and then centrifuge again at 4 °C and 8000 rpm for 20 minutes to collect the precipitate. Dissolve the precipitate in water at a solid-liquid ratio of 1:4, load it into a dialysis bag (molecular weight cut-off 8000 - 14000 Da), and dialyze with ultrapure water at 4 °C for 2 days to desalt, changing the water every 4 hours. Finally, freeze-dry the dialysis solution under the conditions of a cold trap temperature of -45 to -50 °C, a pressure of 30 - 100 Pa, and a freezing time of 60 - 72 h to obtain walnut protein powder (WP).

[0037] (2) Preparation of walnut peptide: Mix walnut protein powder (WP) and deionized water at a ratio of 1:8 (w / w) and stir evenly (magnetic stirring). After pre-treatment in a water bath at 90 °C for 30 min, at pH 7.5 and 50 °C, add plant protease (Nanning Pangbo Bioengineering Co., Ltd.) and trypsin (Nanning Pangbo Bioengineering Co., Ltd.) at 0.6% (w / w) of the mass of walnut protein powder respectively, and enzymatically hydrolyze for 12 h. Treat the enzymatic hydrolysate at 95 °C for 10 min to inactivate the enzyme. After cooling to room temperature, immediately centrifuge the enzymatic hydrolysate (8000 rpm, 4 °C) for 20 min. Then collect the supernatant, and freeze-dry it under the conditions of a cold trap temperature of -45 to -50 °C, a pressure of 30 - 100 Pa, and a freezing time of 60 - 72 h to obtain walnut protein peptide (WPH).

[0038] (3) The 18M_WPH group of mice was intragastrically administered walnut protein peptide at a dose of 666 mg / kg bw for 35 days. On the 7th and 35th days, the Morris water maze and Barnes maze experiments were used to evaluate the effect of walnut peptide on the spatial memory ability of mice. On the 35th day, the feces of the mice were collected for intestinal flora analysis to analyze the changes in the α and β diversities of the intestinal flora, the ratio of Firmicutes to Bacteroidetes (F / B) at the phylum level, and the differential genera at the genus level.

[0039] Comparative Example 1

[0040] A preparation method of walnut protein powder

[0041] (1) Extraction of walnut protein powder: Walnut protein was extracted from walnut meal by the alkali-solution acid-precipitation method. First, the walnut meal was ground into powder, mixed evenly with 8 times its mass of deionized water, and the pH was adjusted to 8.0, followed by stirring at room temperature for 4 hours. Then, centrifugation was carried out at 4°C and 8000 rpm for 20 minutes, and the supernatant was collected and the pH was adjusted to 4.5. After continuous stirring at room temperature for 4 hours, centrifugation was carried out again at 4°C and 8000 rpm for 20 minutes to collect the precipitate. The precipitate was dissolved in water at a solid-liquid ratio of 1:4, loaded into a dialysis bag (cut-off molecular weight 8000 - 14000 Da), and dialyzed against ultrapure water at 4°C for 2 days to desalt, with the water being changed every 4 hours. Finally, the dialysate was freeze-dried under the conditions of a cold trap temperature of -45 to -50°C, a pressure of 30 - 100 Pa, and a freezing time of 60 - 72 h to obtain walnut protein powder (WP).

[0042] Effect Example 1

[0043] Animal administration and grouping: The mice were divided into a 2M group (2-month-old mice, n = 10), a 10M group (10-month-old mice, n = 10), an 18M_Con group (18-month-old mice, n = 10), an 18M_WP group (18-month-old mice intragastrically administered walnut protein, n = 10), and an 18M_WPH group (18-month-old mice intragastrically administered walnut peptide, n = 10). The mice in the 18M_WP group and 18M_WPH group were intragastrically administered at a dose of 666 mg / kg bw every day, and were given walnut protein powder and walnut peptide respectively for 35 consecutive days.

[0044] 1. Effects of natural aging on the cognitive function of mice

[0045] As age increases, cognitive impairment becomes more prevalent. There is a certain correspondence between the age of mice in months and the age of humans. Therefore, we selected mice of different months of age to explore the effect of aging on spatial memory ability. Two-month-old mice correspond to the youth period of humans, when brain function is at its peak of development; 10-month-old mice are equivalent to the middle age of humans, when brain function begins to gradually decline, but still maintains good cognitive ability; 18-month-old mice correspond to the old age of humans, showing obvious cognitive function decline.

[0046] To evaluate the effect of natural aging on the spatial memory ability of mice, the mice were under normal diet management, and the Morris water maze and Barnes maze tests were used to evaluate the effect of natural aging on the spatial memory ability of mice. On the 35th day, mouse feces were collected for intestinal flora analysis to analyze the changes in intestinal flora α and β diversity, the ratio of Firmicutes and Bacteroidetes at the phylum level (F / B), and the differential bacterial genera at the genus level.

[0047] Morris water maze test: The water maze test was carried out in a circular pool with a diameter of 120 cm and a height of 35 cm. Before the test, each mouse received 3 training sessions, during which it was placed in one of the four quadrants. Each test included at least three quadrants, and the mouse could swim for 120 seconds to find the hidden underwater platform. If the platform could not be found, the mouse would be guided to the platform and stay there for 20 seconds to familiarize itself with the environment. The movement trajectory of the mouse, the time to reach the platform (escape latency), the moving distance in the target quadrant, and the number of times crossing the platform were recorded, and these data were used to evaluate the degree of memory consolidation.

[0048] Barnes maze test: The Barnes maze is a classic experimental method used to evaluate the spatial learning and memory ability of mice. The maze consists of a rotatable circular platform with a height of 140 cm and a diameter of 122 cm, surrounded by 19 equally spaced round holes, one of which is connected to the target box and the rest are empty holes. The activity trajectory of the mouse, the time to reach the target box (escape latency), and the number of times exploring the holes were recorded through video detection software. Each mouse had 5 minutes of free exploration time, and after the test, the platform was cleaned and the maze was rotated to eliminate odor interference.

[0049] Analysis of intestinal flora: Mouse fecal samples were collected, and total genomic DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA). The integrity of genomic DNA was detected by agarose gel electrophoresis, and the concentration and purity of genomic DNA were detected by Nanodrop 2000 and Qubit 3.0 spectrophotometers. The V3-V4 hypervariable region of the 16S rRNA gene was amplified using primers 341F (5'-CCTACGGGNGGCWGCAG-3', where W represents A or T) and 805R (5'-GACTACHVGGGTATCTAATCC-3', where H represents A or C or T, and V represents A or C or G), and then sequenced using the Illumina NovaSeq 6000 sequencer. The original read sequences were processed in QIIME2. Adapter and primer sequences were trimmed using the cutadapt plugin. The DADA2 plugin was used for quality control and identification of amplicon sequence variants (ASVs). The ASV representative sequences were classified and assigned using a pre-trained naive Bayes classifier trained on SILVA (version 138.1) with a confidence level of 0.8. For the obtained results, the changes in intestinal flora α and β diversities were analyzed at the overall level; the changes in the F / B value were analyzed at the phylum level; and the differential flora at the genus level was further analyzed.

[0050] The Morris water maze is widely used in cognitive research and can effectively evaluate the short-term and long-term spatial memory of mice. The Barnes maze, by reducing the stress response that may be brought about by the water environment, is more in line with the natural behavioral characteristics of mice and provides another way to evaluate spatial memory.

[0051] 2. Effects of walnut peptides on the cognitive function of mice

[0052] The walnut protein peptide (WPH) obtained in Example 1 was intragastrically administered to the mice in the 18M_WPH group at a dose of 666 mg / kg bw for 35 days. On the 7th and 35th days, the effects of walnut peptides on the spatial memory ability of mice were evaluated through Morris water maze and Barnes maze experiments. On the 35th day, the feces of the mice were collected for intestinal flora analysis. The experimental evaluation method was the same as in step 1.

[0053] The walnut protein powder (WP) obtained from Comparative Example 1 was intragastrically administered to the mice in the 18M_WP group at a dose of 666 mg / kg bw for 35 days. On the 7th day and the 35th day, the Morris water maze and Barnes maze tests were used to evaluate the effect of the walnut protein powder on the spatial memory ability of the mice. On the 35th day, the feces of the mice were collected for intestinal flora analysis to analyze the changes in intestinal flora diversity, the ratio of Firmicutes to Bacteroidetes (F / B) at the phylum level, and the differential genera at the genus level.

[0054] 3. Results

[0055] (1) Spatial cognitive ability

[0056] The results of the water maze place navigation test are shown in the appendix Figure 1 As shown. Compared with 2-month-old mice, the time for 10-month-old and 18-month-old mice to find the underwater platform increased with increasing age. After treatment with the walnut peptide prepared by the present invention (Example 1), the time for 18-month-old mice to find the platform decreased significantly, and the effect was better than that of treatment with the walnut protein powder (Comparative Example 1). It should be noted that treatment with the walnut peptide for 35 days improved the spatial memory ability of 18-month-old mice more significantly than treatment for 7 days (see appendix Figure 2 ). In addition, in the spatial exploration experiment, with the increase of age, the dwelling time of 10-month-old and 18-month-old mice in the target quadrant and the number of times of crossing the target platform both decreased significantly. After treatment with the walnut peptide (Example 1), the dwelling time of 18-month-old mice in the target quadrant and the number of times of crossing the platform increased, while treatment with the walnut protein powder (Comparative Example 1) did not significantly improve these indicators (see appendix Figure 3 and appendix Figure 4 ). This shows that the walnut peptide has a stronger improvement effect on the spatial memory ability of old mice.

[0057] Similar results were also obtained in the Barnes maze place navigation test. Appendix Figure 5 is a typical representative trajectory diagram of the Barnes maze place navigation. The results show that with the increase of age, the time for 10-month-old and 18-month-old mice to find the target hole increased, and the number of times of exploring the target cave decreased. After treatment with the walnut peptide (Example 1), the time for 18-month-old mice to find the target hole decreased, and the number of times of exploring the target cave increased, and the effect was also better than that of the walnut protein powder treatment group (Comparative Example 1) (see appendix Figures 6 - 7 ).

[0058] In summary, the walnut peptide of the present invention can significantly improve the spatial memory ability and spontaneous activity ability of old mice, and the effect is better than that of the walnut protein powder treatment group.

[0059] (2) Effects on intestinal flora

[0060] Numerous studies have shown that gut microbiota play an important role in memory and cognition. Analysis of the α-diversity index showed that with the increase of age, the gut microbiota diversity of 10-month-old and 18-month-old mice gradually decreased. Treatment with walnut peptides (Example 1) could significantly increase the chao1 index and simpson index, while the improvement effect of the walnut protein powder treatment group on gut diversity was weaker than that of the walnut peptide group. In terms of the Shannon index and Pielou evenness index, the walnut protein treatment group was even slightly worse than the old control group (see Appendix Figure 8 ). NMDS analysis of β-diversity showed that there were significant differences in the microbial composition of different groups. The microbial composition of 2-month-old mice and the walnut peptide treatment group was significantly different from the other three groups, and the microbial composition of the walnut peptide treatment group was closer to that of 2-month-old mice (see Appendix Figure 9 ). At the phylum level, with the increase of age, the ratio of Firmicutes to Bacteroidetes (F / B) gradually increased. Treatment with walnut peptides could reverse this trend, and the effect was better than that of the walnut protein powder treatment group (see Appendix Figure 10 ). The results of LEfSe analysis (LDA > 3) showed that 10 taxa were significantly affected after walnut peptide intervention, and the genus Akkermansia (i.e., Akkermansia muciniphila) had the highest abundance (see Appendix Figure 11 ). Further analysis of the relative abundance of the genus Akkermansia in mice of different groups found that walnut peptides had a significant regulatory effect on this taxon (see Appendix Figure 12 ).

[0061] In summary, the walnut peptides obtained by the preparation method of the present invention play a role in improving memory and cognition by restoring the gut microbiota homeostasis of naturally aged mice, especially by increasing the abundance of Akkermansia muciniphila. Therefore, walnut peptides have the potential to be developed into health foods or drugs for improving cognitive function or regulating gut microbiota.

[0062] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0063] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of walnut peptide in increasing the abundance of Akkermansia muciniphila in intestinal flora, characterized in that: The preparation method of the walnut peptide having the regulating effect of Akkermansia muciniphila comprises the following steps: (1) Extraction of walnut protein powder: Walnut protein powder is extracted from walnut meal by alkali dissolution and acid precipitation method; (2) Preparation of walnut peptide: walnut protein powder and deionized water were stirred and mixed; pre-treated in a water bath at 90°C for 30 min; enzymatic hydrolysis was performed for 12 h at pH 7.5 and 50°C using plant protein hydrolase and pancreatic enzyme; the resulting enzymatic hydrolyzate was treated at 95°C for 10 min to inactivate the enzyme; after the enzymatic hydrolyzate was cooled to room temperature, it was immediately centrifuged for 20 min; the supernatant was collected and freeze-dried to obtain walnut protein peptide.

2. The use of the walnut peptide according to claim 1 in increasing the abundance of Akkermansia muciniphila in intestinal flora, characterized in that: The stirring and mixing in step (2) of the preparation method is magnetic stirring, and the ratio of walnut protein powder to deionized water is 1:

8.

3. The use of the walnut peptide according to claim 1 in increasing the abundance of Akkermansia muciniphila in intestinal flora, characterized in that: In step (2) of the preparation method, the added amount of the plant protein hydrolase and pancreatic enzyme is 0.6% of the mass of the walnut protein powder.

4. The use of the walnut peptide according to claim 1 in increasing the abundance of Akkermansia muciniphila in intestinal flora, characterized in that: The centrifugation parameters of the enzymatic hydrolysate in step (2) of the preparation method are 8000 rpm and 4°C.

5. The use of the walnut peptide according to claim 1 in increasing the abundance of Akkermansia muciniphila in intestinal flora, characterized in that: The freeze-drying parameters in step (2) of the preparation method are a cold trap temperature of -45 to -50°C, a pressure of 30 to 100 Pa, a freezing time of 60 to 72 hours, and a moisture content of the walnut peptide after freeze drying of ≤7%.

6. The use of the walnut peptide according to claim 1 in increasing the abundance of Akkermansia muciniphila in intestinal flora, characterized in that: The step (1) of the preparation method is specifically to grind the walnut meal into powder, add deionized water of 8 times the mass of the walnut meal and mix evenly, adjust the pH to 8.0, and stir at room temperature for 4 hours; then centrifuge at 4°C and 8000rpm for 20 minutes, collect the supernatant and adjust the pH to 4.5, continue to stir at room temperature for 4 hours, and centrifuge again at 4°C and 8000rpm for 20 minutes to collect the precipitate; dissolve the precipitate with water at a solid-liquid ratio of 1:4, put it into a dialysis bag, dialyze with ultrapure water at 4°C for 2 days to desalinate, and change the water every 4 hours; finally, freeze-dry the dialyzate at a cold trap temperature of -45 to -50°C, a pressure of 30-100 Pa, and a freezing time of 60 to 72 hours to obtain the walnut protein powder.

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